1. Field of the Invention
The present invention relates to an electrophoresis apparatus capable of separating components in a sample by electrophoresis, and more particularly, it relates to a multi-capillary electrophoresis apparatus having plural capillaries.
2. Description of the Related Art
DNA sequencing has been conventionally carried out by a slab gel method, in which a gel for separating a sample is sandwiched with two sheets of transparent glass plate, and a voltage is applied to both ends of the glass plates to effect electrophoresis. JP-A-7-301619, JP-A-8-297111 and JP-A-9-288091 disclose techniques for uniformly maintaining the temperature of the gel within the slab.
An electrophoresis apparatus of the slab gel method disclosed in JP-A-7-301619 has two sheets of transparent glass plates with a thermal control plate, a Peltier element attached in contact with the back surface thereof, a heat sink attached to the heat dispersion surface of the Peltier element, and a cooling fan dispersing the heat of the heat sink.
In the technique disclosed in JP-A-8-297111, a glass plate and a member having good heat conductivity are provided with both of them being in close contact with each other, and the periphery of the assembly is surrounded with a thermal insulation material, whereby heat dispersion is prevented, and the temperature of the glass plates is uniformly maintained.
The technique disclosed in JP-A-9-288091 involves a gel, a light transmission flat plate for maintaining the gel, a pair of a temperature adjusting element and a temperature controlling element for controlling the temperature adjusting element arranged on both sides of the flat plate, upper and lower buffer solution vessels in contact with the gel and the flat plate, and electrodes immersed in buffer solutions charged in the buffer solution vessels. The temperature of the buffer solution arbitrarily and precisely controls the temperature of the gel.
In recent years, a capillary electrophoresis apparatus is being widely spread. A temperature controlling technique used for the capillary electrophoresis apparatus has been disclosed in JP-A-9-251000, JP-A-10-206384 and JP-A-11-277125.
In the capillary electrophoresis apparatus, electrophoresis is carried out in a capillary tube. The capillary tube is a thin tube having an inner diameter of several tens of μm, an outer diameter of a hundred of μm, and a length of several hundreds of mm. A gel (separating medium) is charged in the pore of the capillary tube, and a testing sample to be measured is introduced into the gel. A high voltage of about several tens of kV is applied to both ends of the capillary tube to effect electrophoresis of the testing sample. For example, fluorescence excited upon irradiating the testing sample with excitation light is detected with a photodetector to effect analysis of the testing sample. A DNA fragment sample with a primer or a terminator labeled with a fluorescent substance, for example, by using the Sanger-Coulson method, is subjected to electrophoresis, and fluorescence from the sample during the electrophoresis is detected to determine the base sequence of the DNA. Such an apparatus is referred to as a DNA base sequence analysis apparatus (DNA sequencer).
In order to improve reproducibility of measurement on electrophoresis, and to improve separation performance for testing samples, it is necessary that the electrophoresis conditions, such as the applied voltage and the composition of the gel, are maintained at constant values. However, the electrophoresis temperature that provides the optimum separation conditions varies depending on difference in sample to be analyzed and difference in separation method. In order to maintain the temperature of the gel in the capillary container part uniformly at a constant value without depending on the outside air temperature and the positions, a thermostat oven or the like may be used.
As another reason why the temperature control is necessary, there is a demand for improvement in throughput. The separation time is being demanded to be shortened owing to increase of the amount of gene to be analyzed along with technological progress in recent years, and thus, the applied voltage to the capillary is being increased. On another front, the amount of Joule heat produced is increased on applying such a high voltage to increase the temperature of the gel, and thus the temperature control becomes further difficult.
In the technique disclosed in JP-A-9-251000, the side surface of the capillary is covered with a transparent member that is a prescribed material having the substantially same refraction index as the capillary, and a temperature controlling means (such as a Peltier element and a heat sink), which maintains the temperature of the sample in the capillary at a substantially constant value, is provided in contact with the transparent member.
The technique disclosed in JP-A-10-206384 uses an air thermostat oven. As a temperature controlling mechanism for maintaining the container part housing a capillary at a constant temperature, a chamber surrounded with a thermal insulation material, such as expanded polyurethane, and a Peltier element are provided. Electrical heating and electrical cooling are carried out by the Peltier element. A fan for forming forced convection is provided in the chamber. A temperature sensor, such as a platinum resistance and a thermocouple, is provided in the chamber, and an output signal detected by the temperature sensor is subjected to feedback to the Peltier element to carry out PID control, whereby the interior of the electrophoresis chamber is maintained at a constant temperature.
The technique disclosed in JP-A-11-277125 uses the similar air thermostat oven as the foregoing technique, and a fan for circulating the air has an aspiration inlet for aspirating the air in the direction of the rotation axis and a blowing outlet for blowing the air in the radial direction. The characteristic feature of the technique is that the fan is provided in such a manner that the thickness direction of the fan agrees with the thickness direction of the air thermostat oven.
Furthermore, JP-A-2001-99813 discloses a technique, in which air in a thermostat oven is circulated, and a wall of an air circulation channel is formed with a material of good thermal conduction, and JP-A-4-231862 discloses a capillary electrophoresis apparatus, in which a uniform air flow is formed in a thermostat oven, and a heat exchanger is provided on the circulation channel.
The slab gel method, the temperature controlling mechanism for an electrophoresis apparatus of the single capillary method, and the temperature controlling mechanism for an apparatus using the air thermostat oven have been described.
In the technique disclosed in JP-A-7-301619, the Peltier element does not cover the entire surface of the glass plate. Therefore, the region that can be controlled for the temperature thereof is limited to local area. In the technique disclosed in JP-A-8-297111, only the amount of dispersed heat due to the Joule heat caused by electrophoresis is adjusted, but there is a problem in that temperature control is carried out by providing a heat source for heating.
Such a technique is demanded in recent years that electrophoresis is carried out by using plural capillaries simultaneously to improve processing performance. In the techniques disclosed in JP-A-9-288091 and JP-A-9-251000, there is limitation in increasing the number of capillaries, and the number of samples that can be simultaneously analyzed is limited. In other words, there is a problem on applying the technique of sandwiching capillaries with plates of plane heat sources to a multi-capillary electrophoresis apparatus that analyzes plural samples at the same time.
A commercially available microtiter plate has 96 holes (hereinafter referred to as a “well”). In the case where samples to be analyzed are set in the wells of the microtiter plate, 96 wells are arranged in a matrix form of 8 rows and 12 columns. It is general that, in the samples put in the wells arranged in the matrix form, the samples in one row among the 8 rows or the samples in one column among the 12 columns are simultaneously analyzed. According to the operation, a bundle of the plural capillaries (hereinafter referred to as a “capillary array” or a “multi-capillary array”), to which the samples are introduced for carrying out electrophoresis, can be arranged in one plane. Therefore, the temperature control can be carried out by applying the technique of sandwiching the capillaries with plates of plane heat sources.
However, in the case where more than 12 samples are necessarily analyzed at the same time, or in the case where all the samples set in the microtiter plate having 96 wells are necessarily introduced to a capillary array and subjected to electrophoresis, 12 wells in one row in the microtiter plate are insufficient, and therefore, the samples in several rows or several columns are simultaneously introduced in the capillary array for analysis. It is impossible in this case to arrange the sampling sections on one plane. Therefore, it is impossible to employ the technique of sandwiching capillaries with plates of plane heat sources, but it is necessary to carry out temperature control by arranging the capillary array in a steric space, such as a thermostat oven.
Joule heat is formed from the gel itself upon electrophoresis. When the number of capillaries becomes larger, the influence of the Joule heat cannot be negligible. In the presence of a heat generating body, the heat transfer coefficient varies due to differences of the flowing direction and the flowing velocity of the air around the body. Therefore, there is such a problem upon using the ordinary air circulation thermostat oven that the temperature of the capillaries is differentiated with respect to the positions.
In the technique disclosed in JP-A-10-206384, the fan arranged inside the thermostat oven is positioned at the substantially central part of the thermostat oven. Therefore, it cannot provide a uniform air flow velocity distribution in the thermostat oven for forming a uniform temperature distribution by forced convection.
The technique disclosed in JP-A-11-277125 uses a microtiter plate. It is necessary to increase the thickness of the thermostat oven in the case where the number of capillaries that are simultaneously used is increased. Therefore, the radiation heat from the plates having good thermal conduction is insufficient in the plane air circulation method, and thus there is a problem in that the temperature difference occurs with respect to the positions.